A long standing problem in light microscopy is how to generate real time stereoscopic images at high magnifications. At low magnifications, where it is simple to provide appropriate left eye and right images for improved depth discrimination, stereoscopic visualization is almost always the preferred imaging mode in applications such as dissection, microsurgery and microassembly. However at higher magnifications, where such depth discrimination could be just as useful in some applications, stereoscopic imaging is very rarely used, in part because of the unavailability of a simple method that does not add a considerable delay to the imaging process. One partial solution has been to occlude opposite halves of the exit pupils of the left and right eyepieces, which creates an illusion of stereopsis, but with a resulting reduced resolution, and no solution of the depth-of-field problem. Another proposed solution has involved projecting the primary real image of a high power microscope onto a surface that was rapidly scanned in depth, and to visualize the light reflected from that surface with a low power dissecting microscope. This caused a substantial light loss, image degradation, and a magnification that changed with depth. Proposals have been made for real time, high magnification stereoscopic visualization, for example by introducing deliberate longitudinal chromatic aberration during confocal imaging, and applying opposite lateral chromatic dispersion to the images for the left and right eyes (Baer, U.S. Pat. No. 3,547,512), however this process cannot work with fluorescence imaging or with monochromatic laser illumination. One technique for producing stereoscopic image pairs that works with confocal imaging is to create a stack of images at different, closely spaced depths, and make two stacks of summated images, one for the right eye where before summation, each image is shifted lateral in one direction by a distance proportional to depth, and this lateral shift is in the other direction for the other eye. Though confocal microscopes frequently have the capability to perform this operation, it is rarely used, partly because it generally introduces an unacceptable delay between exposure and image display.
The problem of producing real time stereoscopic images is particularly great at the cutting edge of high resolution light microscopy using techniques that reduce the diameter of a scanned spot by eliminating fluorescence at the periphery of the spot before significant fluorescent emission (Hell and Wichmann, U.S. Pat. No. 5,731,588, Baer, U.S. Pat. Nos. 5,777,342, 5,866,911, 5,952,668 and 6,259,104 and 6,903,347 which are incorporated herein by reference.). In practice, these techniques have been found to produce photobleaching, which has limited the exposure possible for a specimen, so that techniques requiring many exposures are difficult to implement. Alternative techniques that reduce the size of an excited or excitable spot by using special properties of dyes and proteins that can optically switch between fluorescent and non fluorescent states require long dwell times per pixel, again making producing of many images from a specimen difficult.
Providing a microscope, which combined real time high depth-of-field imaging and stereoscopic visualization with resolution beyond the Abbe limit has remained a long standing unfulfilled need.